Anions play essential roles in biological processes; indeed, it is believed that they participate in 70% of all enzymatic reactions. There is, therefore, intense effort being devoted to the problem of anion complexation and recognition. In the molecular recognition arena, a number of research groups have followed Nature's lead and have designed and synthesized receptors that use hydrogen bonds alone, or in concert with electrostatic interactions, to coordinate to anions. Nonetheless, there remains at present a critical need for additional anion complexing agents that are either easy to make or inherently selective in their substrate binding properties.
Additionally, the separation of anionic mixtures using chromatographic techniques involving anion binding interactions is a little studied area of chemistry. Current techniques for purification of anions such as oligonucleotide fragments, polyphosphate-containing molecules such as ATP, ADP and AMP, carboxylates, or N-protected amino acids involve derivatization prior to separation, leading to decreased yields and cumbersome methodology, or involve a salts separation step following chromatography. This is a very important area to both scientists and clinicians as both mono- and di-nucleotides, natural and synthetic oligonucleotides play critical roles in modern biotechnology as well as medicine. Oligonucleotides are used, for instance, as hybridization probes in blot analyses, primers for PCR amplification, and for site-specific mutagenesis. Furthermore, in some areas, oligonucleotide-derived products are currently being used as probes for the detection of genetic diseases and for proviral HIV, the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Oligonucleotides are also being considered as potential chemotheraputic agents, both directly, i.e., in gene therapy, and in an antisense fashion.
The above-mentioned applications require oligonucleotide materials of impeccable purity (often greater that >99.99%). Such purity, however, is not readily obtained using existing technology. Presently, gene products and other oligonucleotide-type materials are purified using polyacrylamide gel electrophoresis (PAGE). This approach suffers from the requirement of using toxic materials and is painfully manpower-demanding and low-yielding.
Liquid chromatographic techniques, in particular, high performance liquid chromatography (HPLC) and High Performance Affinity Chromatography (HPAC) employing speciality silica gels are currently used to separate biological molecules. Indeed, silica gel phases with bonded groups, such as linear hydrocarbons, amino groups, cyano-groups, carboxylic amides and amino acids, are all known. Unfortunately, few, if any, of these phases are efficacious for the efficient, high-yielding separation of nucleotides and oligonucleotides. Those that work best for this purpose are ion exchange columns which, on a limited basis, can sometimes separate oligonucleotides containing 40 or fewer residues. However, this technique still suffers from several limitations, including the requirement for severe conditions, such as elution at pH 2.7, for routine operation. The use of high concentration buffers (greater than 1M) and gradients that often include formic acid or formamide, also limits the half-lives of ion exchange columns. Reverse phase columns use column media, such as silica gel with appended groups such as alkyl chains, that separate species on the basis of hydrophobic effects. Reverse phase columns may be used at pressures up to 5000 psi. However, in order to separate species such as oligonucleotides on this type of column, protecting groups must first be appended to the oligonucleotide, and ion-pairing reagents must be used, requiring an additional purification step after the chromatographic separation.
A type of stationary phase has been previously described in which purine and pyrimidine bases are bonded to silica gel. With this support, base-pairing interactions between the nucleic acid base pairs and the modified silica gel were expected to improve the resolution obtained in nucleic acid separation procedures. Although such stationary phases were used to separate nucleic acid-free bases, purine alkaloids, nucleosides, and mono- and oligonucleotides, this approach unfortunately has demonstrated the least success in the case of oligonucleotide separation, which is the most important area. Thus, prior to the present invention, there remained a critical need for improved solid supports that would effectively separate nucleotides and oligonucleotides.
Additionally, sapphyrin (an expanded porphyrin known to bind anions) has been attached to a functionalized silica gel. The separatory properties of this material were not sufficient to separate oligonucleotides successfully due to broad peaks observed for (3- to 9-mer) oligonucleotide mixtures. Furthermore, synthetic challenges are associated with preparing the requisite functionalized sapphyrins since they require over 20 synthetic steps to produce.
Current technology for dialysis in medical applications relies on membranes, such as microfiltering cellophane, to filter anions such as chloride anion or phosphate-containing anions from the blood stream. Aluminum hydroxide or calcium carbonate cocktails must be consumed by the dialysis patient in order to bind the anionic species. A major drawback of this technology is that aluminum builds up in cellular membranes to toxic levels over time causing ailments including dementia and death. Calcium carbonate offers a less toxic substitute, however, it is less efficient and is associated with hypercalcemia.
Water-soluble anion binding agents are desired as drug delivery agents. For examples many anti-viral drugs only show activity when phosphorylated. However, many phosphorylated drug derivatives are too polar to pass through cell wall membranes. A water-soluble anion binding agent may be able to encapsulate the negative charge and so allow the drug to pass through cell walls.
The synthesis of new molecular devices designed to sense and report the presence of a particular substrate is an area of analytical chemistry that is attracting attention. The detection of anionic species is a particular challenge, as anions are difficult to bind and are generally larger than cations leading to a smaller charge-to-radius ratio.
Molecular recognition of neutral compounds presents a challenge in the area of supramolecular chemistry. Binding of substrates, such as short-chain alcohols and simple monoamides, is particularly difficult because these molecules have few functionalized sites available for hydrogen bonding, and they lack the large hydrocarbon surfaces necessary to participate in efficient hydrophobic or π-π stacking interactions. Association constants for neutral substrate-synthetic receptor complexes are thus generally modest, even though the architectural complexity of the receptors is often high.
Cation binding agents may be useful as sensors for particular cations or as sequestering agents. Additionally, particular cation-complexes may be useful in medicine as imaging agents or in the treatment of disease.
One aspect of the present invention involves calixpyrroles. Calixpyrroles represent a subset of a class of macrocycles that was previously termed porphyrinogens. Porphyrinogens are non-conjugated macrocyclic species composed of four pyrrole rings linked in the α-position via sp3 hybridized carbon atoms. Porphyrinogens that carry meso-hydrogen atoms are prone to oxidation to the corresponding porphyrins. Fully meso-non-hydrogen-substituted porphyrinogens are generally stable crystalline materials. The first such macrocycle, meso-octamethylcalix[4]pyrrole, was reported over a century ago by Baeyer (Ber. Dtsch. Chem. Ges. 1886, 19, 2184) using a condensation between acetone and pyrrole catalyzed by hydrochloric acid, however, the structure of the molecule was not elucidated. This method was reportedly refined by Dennstedt and Zimmerman (Ber. Dtsch. Chem. Ges. 1887, 20, 850) by replacing the hydrochloric acid catalyst with “chlorzink” (presumably zinc chloride) and heating the reaction. Chelintzev and Tronov reportedly produced calix[4]pyrroles by the method of condensing acetone and pyrrole, methylethyl ketone and pyrrole, methylhexylketone and pyrrole and a mixture of acetone and methylethylketone with pyrrole (J. Russ. Phys. Chem. Soc. 1916, 48, 1197; Chem Abstr. 1917, 11, 1418). Chenlintzev, Tronov and Karmanov reported further production of calixpyrroles by condensing cyclohexanone with pyrrole and a mixture of acetone and cyclohexanone with pyrrole (J. Russ. Phys. Chem. Soc. 1916, 48, 1210). Rothemund and Gage reportedly refined Dennstedt and Zimmerman's method by replacing the acid catalyst with methanesulfonic acid (J. Am. Chem. Soc. 1955, 77, 3340). Brown, Hutchinson and MacKinnon (Can. J. Chem. 1971, 49, 4017) reportedly repeated the synthesis of meso-tetracyclohexylcalix[4]pyrrole and assigned a tetrameric macrocyclic structure. J.-M. Lehn and co-workers have reportedly synthesized meso-octa-3-chloropropylcalix[4]pyrrole by an unpublished procedure and converted it to meso-octa-3-cyanopropylcalix[4]pyrrole (B. Dietrich, P. Viout and J.-M. Lehn in Macrocyclic Chemistry, VCH Publishers, Weinheim, 1993, pg 82). The metal cation binding of deprotonated calix[4]pyrrole macrocycles has been studied by Floriani and co-workers (Chem. Commun. 1996, 1257). Floriani has reportedly developed a method for expanding the pyrrole rings of metal-bound deprotonated calix[4]pyrroles forming calix[1]pyridino[3]pyrroles and calix[2]pyridino[2]pyrroles (J. Am. Chem. Soc. 1995, 117, 2793). A further prior art method reports using pyrrole, a C4–C6 saturated alicyclic ketone and an acid containing vinyl groups or triple bonds to form a polymerized resin (WO 93/13150). In this case, the resulting products are undefined since it appears to be unknown where the modifying group is attached to the product.
While the term porphyrinogen is now widely accepted in the literature, the present inventors believe the fully meso-non-hydrogen-substituted systems are misnamed. They are not bona fide precursors of the porphyrins and might, therefore, be better considered as being calixpyrroles rather than porphyrinogens. Such a renaming has precedent in the chemistry of other heterocyclic ring systems. Interestingly, while related functionalized calixarenes, a class of molecules containing phenol (as opposed to pyrrole) subunits have been shown to be capable of binding anions, unmodified calixarene frameworks show no affinity for anionic guests, demonstrating that the anion-binding properties are not an inherent part of the calixarene ring structure.
While a few other fully meso-non-hydrogen-substituted porphyrinogens are known, no one prior to the present inventors recognized their properties and consequent uses as described herein.